1. Field of the Invention
This invention relates to an ultra-minute microscope for spectroscopy enabling emission, scattering and absorption spectroscopy by use of a high-resolution wavelength-variable laser and more particularly to an ultra-minute microscope for spectroscopy enabling real-time observational spectroscopy on functional dye molecules, light-emitting centers, quantum dots, quantum wires and other single-quantum structures and molecules in solution, chemical reactions, genes, proteins, cells and so on.
2. Description of the Prior Art
Although single-molecule spectroscopy is a new science whose theoretical possibility was verified only several years ago, its extremely powerful spectroscopic capability is already contributing to steady elucidation of the fundamentals of micro-scale interactions. However, the spectroscopic sensitivity is still insufficient owing to the weak signal strength and measurement instrument technologies. The kinds of molecules that can be detected therefore remains severely limited.
Single-molecule spectroscopy involves observation of the fluorescence excitation spectrum from individual molecules embedded in a solid substance when the molecules are excited at cryogenic temperatures by a wavelength-variable laser exhibiting high monochromaticity. An essential requirement for this observation is that the number of molecules excited by the laser beam be reduced to the absolute minimum. This is achieved by attaching the end face of a single-mode optical fiber directly to the sample, focusing the excitation light by use of a small lens, or placing the sample behind a pinhole.
In addition, the density of the sample has to be diluted to at least around 10.sup.-7 to 10.sup.-8 mole/liter. The optical system collecting light emitted from a single molecule generally uses a parabolic mirror, an elliptical mirror, or a microscope object lens. These components are large and tend not to be so efficient in actual application. Sample mounting requires a high degree of skill, while handling requires meticulous care and is highly troublesome.
In order to overcome these problems of the prior art, the inventors earlier developed an ultra-minute light collecting system for spectroscopy that, in essence, uses a gradient index rod-shaped micro-lens to reduce the number of reflecting mirrors and optical components at the interface, eliminate loss caused by the presence of the multiple lens surfaces of an objective lens or the like and further enhance signal light collection efficiency by directly using the end face of the rod-shaped lens as the sample substrate, thereby basically enabling integration of all required functions in a single optical element (see Japanese Patent Public Disclosure Hei 10-62347).
The performance of this light collecting system was compared with that of existing technologies. Molecules of terrylene, a conjugated polycyclic compound excellent in light emission efficiency, were selected as the molecular species for spectroscopy. The sample was prepared by dispersion-doping the terrylene molecules into a crystalline medium consisting of linear chain alkane molecules (i.e., Shpolskii medium). A thin film of tungsten was vapor-deposited on one end surface of a 0.25-pitch, 1.8 mm-diameter rod-shaped lens and an approximately 4 .mu.m-diameter hole was formed at the center of the film. The sample was packed into the hole and spectroscopy was conducted at a temperature of 1.7K. Good emission spectrum signals were obtained from the single molecules.
The spectrum broadening behavior due to saturation with increasing excitation light intensity was quantitatively measured and compared with the results obtained with other experiments. The good agreement warranted the conclusion that the spectral signals were signals from single molecules. The level of the background noise was approximately the same as that in existing methods but the excitation light intensity that gave about the same level of signal was in effect markedly lower than by the existing light-collecting systems, eventually improving a signal-to-noise (S/N) ratio greatly. This demonstrated the potential of the light collecting system to provide a marked efficiency improvement.
In this earlier developed light collecting system, the minute space for accommodating the sample was, as explained above, secured by providing the thin metal film coating and forming at the center thereof a pinhole of several micrometers in diameter. It is otherwise possible to bring a single-mode optical fiber close to the center of the rod-shaped lens surface, utilize the intervening gap and use the minute space formed by the single-mode optical fiber core as the minute space for holding the sample. However, these methods of securing the minute space are disadvantageous in that they hinder efficient use of the light collecting system. They also make it impossible to acquire 2-D, 3-D nor real-time images.
An object of this invention is therefore to overcome the foregoing disadvantages by providing an ultra-minute microscope for spectroscopy that can be operated with high efficiency and can provide 2-D, 3-D and real-time images.